Juvenile hormone (JH) is a sesquiterpenoid produced in specialized endocrine glands, the corpora allata (CA). JH controls metamorphosis and reproduction in insects and this endocrine system can be exploited as a target for agents active against insect pests of public health importance (e.g. the JH analog medioprene). The long term objective of this proposal is to gain a better understanding of the biochemical and neuroendocrine regulation of JH synthesis in insects. This research will facilitate the rational design of compounds that disrupt JH synthesis. The present project will focus on the viviparous cockroach, Diploptera punctata, an insect that produces a single JH homolog (JH III) at very high rates in a well-defined physiological context. Several allatostatins, peptides that inhibit JH synthesis, have been isolated from the brain of D. punctata and sequenced over the last few years. Work on the biochemistry of JH synthesis and its neuroendocrine control in adult female D. punctata will be continued with the following specific aims: 1/ To clone and sequence the allatostatin gene(s) from D. punctata. The allatostatin gene(s) will be cloned from a brain cDNA library or a genomic library using oligonucleotide screening, antibody screening or PCR. The number and structure of the allatostatin gene(s) will explain the basis of the diversity of allatostatic peptides and facilitate the characterization of allatostatin-related peptides occurring naturally in this species. 2/ To characterize the allatostatins gene product(s). The putative peptide products of the allatostatin gene(s) will be compared to known peptides and new peptides will be synthesized and tested for inhibitory activity on the corpora allata. Isolation and purification of these new peptides from brain extracts will be facilitated by an allatostatin ELISA. 3/ To study the physiology of allatostatins. With the help of recently developed ELISAs for allatostatins and chromatographic separation as needed, the levels of allatostatins will be followed in adult females. Particular attention will be devoted to the respective ratio of peptides in the hemolymph, in different tissues and at different times during a cycle of JH synthesis. Allatostatins will be tested in bioassays for salivary gland secretion and hindgut myotropic activity to establish whether they have a physiological function in peripheral tissues, as suggested by immunolocalization studies. New bioassays may facilitate future structure/activity studies on these peptides. 4/ To study the acquisition of allatostatin sensitivity by the CA in vitro. The rapid increase in allatostatin sensitivity observed at the end of vitellogenesis in vivo will be studied with an in vitro assay, and the factor(s) responsible for this change will be characterized.